1. Field of the Invention
The present invention relates to a method for the production of methionine using modified strains with attenuated transformation of threonine. This can be achieved by reducing threonine degradation to glycine, and/or by reducing its transformation to α-ketobutyrate. The invention also concerns the modified strains with attenuated transformation of threonine.
2. Description of Related Art
Sulphur-containing compounds such as cysteine, homocysteine, methionine or S-adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals. In particular methionine, an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Nevertheless most of the methionine that is produced is added to animal feed.
With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Chemically D,L-methionine is commonly produced from acrolein, methyl mercaptan and hydrogen cyanide. Nevertheless the racemic mixture does not perform as well as pure L-methionine, as for example in chicken feed additives (Saunderson C. L., (1985) British Journal of Nutrition 54, 621-633). Pure L-methionine can be produced from racemic methionine e.g. through the acylase treatment of N-acetyl-D,L-methionine which increases production costs dramatically. The increasing demand for pure L-methionine coupled to environmental concerns render microbial production of methionine attractive.
Methionine biosynthesis depends on homoserine, cysteine and C1 unit productions. Homoserine is a derivative of aspartarte and provides the carbon skeleton for methionine. Homoserine can also be transformed into threonine, which in turn is the precursor of (i) isoleucine and which can also be transformed into glycine (ii). These two reactions consume threonine and draw more homoserine into the threonine pathway, thus reducing the flux into the methionine pathway.
(i) For the production of isoleucine, threonine is deaminated to a-ketobutyrate, a reaction catalysed by threonine deaminase or threonine dehydratase encoded by the genes ilvA (EC 4.3.1.19, Ramakrishnan et al., 1965, J Bacteriol, 89:661) and tdcB (EC 4.3.1.19, Goss et al., 1988, J Bacteriol, 170:5352), respectively. Serine deaminases encoded by sdaA (EC 4.3.1.17; Su et al. 1989, J Bacteriol, 171:5095; SEQ ID NO: 21) and sdaB (EC 4.3.1.17, Su and Newman, 1991, 173:2473) are also known to encode some threonine deaminase activity.
(ii) Two pathways for threonine glycine transformation are present in E. coli: 
(A) Threonine can be transformed into glycine by two consecutive reactions catalyzed by threonine dehydrogenase (Tdh; E.C. 1.1.1.103) and 2-amino 3-keto butyrate-coA-lyase (Kbl; E.C. 2.3.1.29) (Boylan S. A. et al., 1981, Journal of Biological Chemistry, 256, 4, pp 1809-1815; Mukherjee J. J. et al., 1987, Journal of Biological Chemistry, 262, 30, pp 14441-14447). These reactions generate a molecule of acetyl-coA and NADH each. (Komatsubara S. et al., 1978, Journal of Bacteriology, 1981, 135, pp 318-323).
(B) Threonine can also be transformed into glycine directly via a retroaldol mechanism catalysed by threonine aldolase. This reaction generates an acetaldehyde and a glycine (Plamann M. D. et al., 1983, Gene, 22, 1, pp 9-18). Threonine aldolase activity carrying enzymes in E. coli are encoded by the following genes: ltaE (Liu J L et al., 1998, European Journal of Biochemistry, 255, 1, pp 220-226), kbl (Markus J. P. et al., 1993, Biochemica et Biophysica Acta, 1164, pp 299-304) and glyA (Schirch V. et al., 1968, Journal of Biological Chemistry, 243, pp 5561; Schirch V. et al., 1985, Journal of Bacteriology, 163, 1, pp 1-7).
LtaE is a low specific threonine aldolase which is thought to be involved in the degradation of threonine to form acetaldehyde and glycine (Liu J L et al., 1998, European Journal of Biochemistry, 255, 1, pp 220-226).
The primary activity of Kbl is 2-amino-3-ketobutyrate CoA ligase (E.C 2.3.1.29), which consists of the deacetylation of the 2-amino 3-ketobutyrate to form glycine and acetyl-coenzyme A (Mukherjee J.J. et al., 1987, Journal of Biological Chemistry, 262, 30pp 14441 - 14447). It has been shown to posses TAL activity, which makes it a versatile enzyme for threonine degradation (Markus J.P. et al., 1993, Biochemica et Biophysica Acta, 1164, pp 299-304).
The primary activity of GlyA (SEQ ID NO: 22) is serine hydroxymethyltransferase (SHMT) (E.C. 2.1.2.1). It catalyses the conversion of the amino acid serine and tetrahydrofolate (THF) into glycine and 5,10methylene-THF (for review: Schirch V. et al., 2005, Current opinion in Chemical biology, 9, pp 482-487). Among the other secondary activities catalyzed by GlyA (for review: Schirch L., 2006, Advances in enzymology and related areas of molecular biology, 53, pp 83-112), only threonine aldolase (TAL) seems to be physiologically relevant. GlyA was crystallized (Scarsdale et al., 2000, Journal of Molecular Biology, 296, pp 155-168) and studies have been done to elucidate the origin of the substrate specificity (Angelaccio S. et al., 1992, Biochemistry, 31, pp 155-162). Angelaccio et al. mutated all threonines in the vicinity of the active site to alanine, and noticed that mutation T226A increased the Km of threonine by a factor 1.8 and decreased TAL activity to levels inferior to their quantification limit, while not modifying the Km for THF nor the Km for serine. Nevertheless, the mutation has a strong impact on the SHMT activity, since the kcat for the SHMT reaction was decreased by a factor 32.
Strains for producing methionine being modified for an improved yield are now extensively disclosed in the art. It is now understood that the methionine biosynthesis pathway is particularly complex with genes involved in many other pathways. Therefore, enhancing or attenuating a gene susceptible to be beneficial to promote the synthesis of methionine at first sight may end into an opposite result. It is known that genes involved in threonine consumption are also known to be involved in C1 production.
The inhibition of the activity or the attenuation of the expression of proteins involved in threonine degradation are already disclosed in several works. Simic et al. (Simic et al, 2002, Applied and Environmental microbiology, 68(7), pp 3321-3327) disclose attenuation of aldole cleavage of threonine activity of the GlyA protein to enhance threonine production in Corynebacterium glutamicum. Martinez-Force et al. (Martinez-Force et al, 1994, Biotechnology Progress, 10(4), pp 372-376) disclose deletion of the ilv1 gene in Saccharomyces cerevisiae to enhance threonine production. Moreover, in this study, the authors show that there is no correlation between threonine and methionine accumulation and the decrease in threonine deaminase activity. Liu et al. (Liu et al, 1998, European Journal of Biochemistry, 255(1), pp 220-226) detailed the characterization of the LtaE enzyme from Escherichia coli and its role in the growth of cell. Finally, Lee et al. (Lee et al, 2007, Molecular Systems Biology, 3(149), pp 1-8) disclose deletion of tdh and mutation of ilvA genes to enhance threonine production.
All of these studies are exclusively oriented towards threonine production. It has never been considered in the prior art to prevent threonine consumption as a solution to improve methionine biosynthesis.
Despite the complexity of the methionine metabolic pathway, the inventors have found a method to increase methionine production by acting on the threonine transformation.